Intracameral Injection in Rats with Low Risk of Adverse Effects

Intracameral injections allow to bypass the outer ocular barrier and directly deliver compounds to the aqueous humour. However, conducting intracameral injection in rodent present technical challenges that may result in adverse effect and impact experimental outcome. We therefore design the protocol considering the rats's eye anatomy to allow intracameral injection with minimal risk of adverse effects.

Intracameral injections in rodents involve a risk of tissue damage due to the small eye dimensions, limited aqueous humour volume and lens position. Tissue damage during manipulation can leads to complication like anterior chamber shallowing, lens injury, and anterior cell damage and inflammation. All of this cause experimental variability and influence findings.

The length and angle of the incision are critical. We make the incision in the central corneal area where the anterior chamber is the deepest, at an angle close to flat. This generates a long tunnel, which helps in reducing the loss of aqueous humour.

Injecting through the tunnel improves precision and reduces the likelihood of touching the lens. The described intercom injection method can be utilized in various experimental settings to generate disease models or assess different treatments. We expect that the precision and reproducibility of this injection technique will be a valuable tool in experimental ophthalmology.

To begin, prepare the animal for surgery and apply 0.3%ofloxacin and 0.1%dexamethasone to the animal's eye. Using surgical ophthalmic forceps, hold the superior sclera at the vertical midline next to the corneoscleral junction to stabilize the eye. Under a surgical microscope, place a sterile 0.8 millimeter 31 gauge stiletto blade in the paracentral corneal region on the vertical midline.

In this position, puncture the cornea to make an incision and create a long tunnel until it penetrates into the central area of the anterior chamber. Then, apply topical 0.3%ofloxacin and 0.1%dexamethasone to the eye. Next, under a slit microscope, observe the depth of the anterior chamber of the injected eye compared to the non injected eye.

Also, observe the lens of the injected eye compared to the non injected eye. Begin by loading five microliters of trypan blue into a sterile 10 microliter glass Hamilton syringe, equipped with a 34 gauge blunt needle. Then, insert the loaded syringe needle through the self-sealing corneal tunnel into the anterior chamber.

Inject the solution in the eye and hold the needle in place for two to three seconds until all fluid clears. Pull out the needle gently. Examine under slit microscopy.

Evaluate the depth of the anterior chamber to exclude shallowing, and verify the presence of trypan blue in the anterior chamber. Using this protocol, the corneas of Sprague Dawley rats were injected with trypan blue. Immediate slit lamp examination post-injection revealed that the anterior chamber was visibly stained with trypan blue, confirming the successful delivery of the substance to the target area.

To begin, load five microliters of hoechst solution into a sterile 10 microliter glass Hamilton syringe attached to a 34 gauge blunt needle. Insert this syringe needle into the anterior chamber through the self-sealing corneal tunnel and inject the solution. Hold the needle in place for two to three seconds until all fluid clears.

Pull out the needle slowly to remove it, avoiding leakage. Next, enucleate both eyes. To isolate the corneas, cut the sclera around the optic nerve and make four additional cuts from the optic nerve towards the cornea.

Drain the excess fluid with the whatman filter paper. Spread out the cut sclera and separate the retina from the sclera. Pull out and discard the lens and retina.

Next, remove the iris. Cut and discard the sclera around the cornea and carefully lift up the cornea. Placing the corneas on a glass slide, stain them with 0.5%alizarin red to identify endothelial cells.

After removing the excess stain, wash the cornea three times and place a cover slip on it. Examine the corneas under a light microscope to image the alizarin red staining of endothelial cells. Also, evaluate the corneas under a fluorescent microscope to observe hoechst staining and compare it to the non injected cornea as control.

Hoechst dye, a DNA binding cell permeable fluorescent marker was utilized to assess drug bioavailability through intracameral injection. Corneal endothelial cell uptake of hoechst was examined 15 post-injection by fluorescent microscopy. Alizarin red staining highlighted the intercellular borders of the endothelial layer.

Also, positive hoechst staining within corneal endothelial cell nuclei confirmed the successful delivery of the dye via this method.